1. Field of the Invention (Technical Field)
The present invention relates to methods and apparatuses for in situ denitrification, most particularly biodentrification.
2. Background Art
Bioremediation is the use of microorganisms to convert harmful chemical compounds to less harmful chemical compounds in order to effect remediation of a contaminated site. The microorganisms are generally bacteria but can be fungi. The contaminants can be organics such as petroleum hydrocarbons and domestic wastewater or inorganics such as nitrate and metal ions. Microbial growth and metabolism require suitable nutrients to construct new cells and materials to supply energy through oxidation-reduction reactions.
The nutrients that are used to construct new cell components include inorganic or organic compounds that provide the major elements (carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, etc.) to the cell. Elements that are not major components of building block material but are still necessary for growth (micro-nutrients) include such elements as Mg, Ca, K, Fe, Mn, Na, Zn, and Cl. Groundwater contaminated with nitrate and sulfate requires addition of only phosphorus nutrient and hydrocarbon substrate for the supply of major elements. While a variety of minerals are required by microorganisms, they are needed in trace amounts, and adequate amounts are normally present in most groundwater.
Microbial growth and metabolism need an energy supply. Microorganisms obtain their energy for metabolism and biosynthesis either by converting sunlight into chemical energy (phototrophs) or extracting energy from organic or inorganic chemicals (chemotrophs). Oxidation-reduction reactions are the basis of all energy-producing reactions, and adenosine triphosphate (ATP) is the principle energy-transport molecule of the cell. Microorganisms then use this energy to perform specific functions, one of which is biosynthesis of new cell components. Energy can be extracted from substrates in one of three ways: respiration, fermentation, and anaerobic respiration. Aerobic bacteria use oxygen as a terminal electron acceptor and oxidize carbon substrate to CO2 (i.e., respiration). Some anaerobic bacteria use inorganic molecules such as NO3xe2x88x92, NO2xe2x88x92, or SO42xe2x88x92 as electron acceptors with CO2 as the final carbon oxidation product (i.e., anaerobic respiration), while others use organic molecules such as pyruvate as an electron acceptor with fermentation products such as lactic acid as a final carbon oxidation product (i.e., fermentation). Facultative bacteria have the capability of growing in the presence or absence of oxygen.
Denitrifying bacteria, which are able to use nitrogen oxides as electron acceptors in place of oxygen, are essentially facultative bacteria. Obligate anaerobes, such as sulfate reducing bacteria often survive in the presence of facultative bacteria. There are two nutritional types of microorganismsxe2x80x94those that obtain their carbon for biosynthetic processes from organic compounds (i.e., heterotrophs) and those that obtain their carbon for biosynthetic processes from CO2 (i.e., autotrophs). Most denitrifying bacteria and sulfate reducing bacteria are heterotrophic and few can grow autotrophically.
Nitrate pollution in groundwater is a common problem in all European and North American countries. Nitrate contamination often exceeds the maximum contaminant limit of 10 mg N/L, and poses a major threat to drinking water supplies. The standard was imposed because nitrate is linked to infant methemoglobinemia (xe2x80x9cblue babyxe2x80x9d syndrome). The formation of nitroso-compounds which are known carcinogens also has been linked to nitrate. Conventional nitrate treatment technologies include reverse osmosis and ion exchange. Activated carbon adsorption in conjunction with pH adjustment has also been used in experimental studies to successfully remove nitrate. Biological denitrification is a technology mainly studied for surface water treatment. Compared to conventional technologies, biological denitrification is very cost-effective and is promising for in situ remediation, particularly as practiced with the present invention.
The main biological processes involving inorganic nitrogen are shown in FIG. 1. Nitrogen fixation involves the synthesis of cellular nitrogen compounds from elementary nitrogen. It is associated primarily with certain agricultural plants in which bacteria in a symbiotic or free living state. Deamination reactions are associated with the lysis of dying cells and the formation of ammonia from organic nitrogen compounds. Nitrification is the oxidation of NH4+ to nitrate, via nitrite, and is carried by nitrifying bacteria. With regard to nitrate metabolism, assimilation is defined as the conversion of nitrate to cellular organic nitrogen via ammonia, and dissimilation (or nitrate respiration) is defined as the oxidation of carbon compounds at the expense of nitrate which acts as the alternative electron acceptor to oxygen.
Denitrification is a special case of dissimilation in which gaseous nitrogens are end products. The principal products are nitrogen gas (N2) and nitrous oxide (N2O), though nitric oxide (NO) has occasionally been detected. During the denitrification process, nitrogen oxides serve as terminal electron acceptors instead of oxygen and are reduced by a unique suite of complex enzymes that conserve energy in several reductive steps by electron transport phosphorylation. The pathway of denitrification is thought to be:
NO3xe2x88x92xe2x86x92NO2xe2x88x92xe2x86x92NO(g)xe2x86x92N2O(g)xe2x86x92N2(g).
The reduction of nitrate to nitrite is known as denitratation, and the reduction of nitrite is called denitritation. Each reaction step involves a different enzyme. For example, nitrate reductase (NaR) catalyzes the reduction of nitrate to nitrite and nitrite reductase (NiR) catalyzes the reduction of nitrite to gaseous products.
Nitrite and N2O are often observed to accumulate temporarily during denitrification. This accumulation can often been explained by relative differences in reaction rates for the different steps in the sequence. For example, when denitritation rate is higher than denitratation rate, nitrite is reduced as soon as it appears and so, it does not accumulate in the system. But if denitratation is faster than denitritation, nitrite build-up will be noticed. Several reasons have been suggested to explain this phenomenon: evolution of the microbial population, enzymatic adaptation to changes in the environment (particularly, dissolved oxygen concentration and pH), inhibition of nitrite reductase, or effect of external carbon loading.
Denitrifying bacteria, which are able to use nitrogen oxides as electron acceptors in place of oxygen with the evolution of gaseous products, are biochemically and taxonomically very diverse. Most bacteria are heterotrophs and some utilize one-carbon compounds, whereas others grow autotrophically on H2 and CO2 or reduced sulfur compounds. One group is photosynthetic. Most have all of the reductases necessary to reduce NO3xe2x88x92 to N2, some lack NO3xe2x88x92 reductase and are termed NO2xe2x88x92 dependent, and others lack N2O reductase and thus yield N2O as the terminal product. Still other organisms possess N2O reductase but cannot produce N2O from NO3xe2x88x92 or NO2xe2x88x92. Among the denitrifying bacteria, the genus Pseudomonas, which includes the most commonly isolated denitrifying bacteria from both soils and aquatic sediments, may represent the most active denitrifying bacteria in natural environments. Some of them are NO2xe2x88x92 dependent, and some strains produce N2O. Denitrifying pseudomonads include P. denitrificans, P. fluorescens, P. stutzeri, P. aerogenes, P. aureofaciens, P. caryophylli, and P. chlororaphis. 
Increasing the population of denitrifying bacteria is the ultimate goal of denitrification. Natural in situ biological denitrification, which is too slow to do efficient groundwater remediation, can be promoted by adding suitable nutrients. Addition of a carbon substrate is required to increase the amount of denitrifying bacteria and, consequently, achieve a satisfactory degree of denitrification for groundwater with a low BOD (Biochemical Oxygen Demand) to nitrogen ratio. Amendment of sulfur and nitrogen is usually not necessary, because they are already typically present in the groundwater (in the form of sulfate and nitrate). Although ammonia (NH3) is the most efficiently used nitrogen source for microbial synthesis since it can be incorporated directly into carbon skeletons to produce amino acids, nitrate and/or nitrite can be assimilated by many denitrifying bacteria to NH3 with electrons supplied by NADPH2 (reduced nicotinamide adenine dinucleotide phosphate). With regard to micro-nutrients, their addition is typically not necessary for in situ bioremediation, and may even have a negative impact on biological activity in soil. But the amendment of phosphorus nutrient is often essential for activating bacterial growth in the groundwater, and is best supplied as in the present invention.
Many organic compounds including carbohydrates, alcohols, organic acids and amino acids can serve as effective carbon sources for heterotrophic denitrifying organisms. Methanol and ethanol are the most commonly used external carbon sources. Besides methanol and ethanol, other carbon sources have been used as electron donors for denitrification, including glucose, sucrose, isopropanol (in practice, isopropanol itself contributes little to denitrification while the converted acetone plays the main role of electron donor), ascorbic acid, lactic acid, acetate, and even hydrogen gas generated through the electrolysis of water. Acetate, lactate, glucose, methanol and ethanol are often employed for denitrification because these chemicals are relatively cheap and commercially available.
Phosphorus is an essential element for bacterial growth. Phosphorus appears in organic molecules primarily as a component of nucleotides such as ATP, which is important as a carrier of energy and phosphate, and as a constituent of nucleic acids. Typical phosphate concentrations in soil ranges from 50 to 5000 mg/L and in groundwater from 100 to 1000 xcexcg/L, which can support fairly high cell density for in situ bioremediation. However, often a phosphorus nutrient has to be added. Consequently, phosphorus amendment for in situ denitrification is crucial to success of many denitrification sites.
To reiterate, bioremediation is driven by increasing the size and mass of microbial populations. Microorganisms must transform environmentally available nutrients to forms what are useful for incorporation into cells and synthesis of cell polymers. Reducing nutrients and synthesizing new cell mass requires energy. This energy is supplied through electron transfer from electron donors to terminal electron acceptors. The terminal electron acceptor used during metabolism is important for establishing the redox conditions and the chemical speciation in the vicinity of the cell. Common terminal electron acceptors include oxygen under aerobic conditions, and nitrate, Mn(IV), Fe(III), sulfate, and carbon dioxide under anaerobic conditions. Although uranium is toxic to microorganisms, reduction of U(VI) is also able to serve as terminal electron acceptor for some bacteria.
Contaminants in groundwater can include nitrate, U(VI), and sulfate. These inorganic chemicals can serve as electron acceptors for bacterial growth in anaerobic conditions. Consequently, the addition of electron donors is required for remediation purposes. Glucose acetate, ethanol, methanol and lactate are possible electron donors. Phosphorus is an essential element for bacterial growth. The amendment of phosphate needs to be employed to achieve appreciable remediation rates.
Microorganisms preferentially utilize electron acceptors that provide the maximum free energy during respiration. Of the common electron acceptors used by microorganisms, oxygen has the highest redox potential and provides the most free energy to microorganisms during electron transfer. The redox potentials of nitrate, U(VI), Fe(III), and sulfate are lower compared to the redox potentials of oxygen. Consequently, they yield less energy during substrate oxidation and electron transfer according to the order as shown in FIG. 2. The calculation of the redox potential data is summarized in Appendix 1 of Lu, xe2x80x9cSequential Bioremediation of Nitrate and Uranium in Contaminated Groundwaterxe2x80x9d, unpublished Ph.D. Dissertation, University of New Mexico (publication anticipated after May 9, 1998), and the entirety of the dissertation is incorporated herein by reference. When amended with carbon substrates (electron donors), the indigenous bacteria (likely a consortium of bacteria with many different species) in the groundwater will consume dissolved oxygen first and then nitrate. Only after all nitrate is consumed, will anaerobic bacteria then begin to grow and use ferric ion (by IRB) or sulfate (by SRB) as alternate electron acceptors. The stepwise reactions are also shown in FIG. 2.
If groundwater is amended with potassium acetate as an carbon substrate (electron donor) along with a phosphorus nutrient, and assuming that acetate can support the growth of denitrifying bacteria, IRB (ion-reducing bacteria) and SRB (sulfate-reducing bacteria), and is oxidized to carbon dioxide, the total reactions being shown in Equations 1-6:
CH3COOxe2x88x92+2O2xe2x86x922HCO3xe2x88x92+H+xe2x80x83xe2x80x83(1)
5CH3COOxe2x88x92+8NO3xe2x88x92+3H+xe2x86x924N2↑+10HCO3xe2x88x92+4H2Oxe2x80x83xe2x80x83(2)
Denitrifying bacteria are facultative, so they can use both dissolved oxygen and nitrate as electron acceptors. Denitrifying bacteria will consume oxygen first and then nitrate. As will be shown in the experimental parts of the dissertation, consumption of oxygen is the prerequisite of denitrification. The redox potential (Eh) value decreases continuously during denitrification, and is lower than xe2x88x92100 mV after the completion of denitrification. Neutral pH and low Eh are two essential growth conditions of IRB and SRB. The stepwise reactions in anaerobic conditions are:
CH3COOxe2x88x92+4UO2(CO3)22xe2x88x92+4H2Oxe2x86x924UO2+10HCO3xe2x88x92+H+xe2x80x83xe2x80x83(3)
CH3COOxe2x88x92+8Fe(OH)3+6HCO3xe2x88x92+7H+xe2x86x928FeCO3+20H2Oxe2x80x83xe2x80x83(4)
CH3COOxe2x88x92+SO42xe2x88x92xe2x86x92HSxe2x88x92+2HCO3xe2x88x92xe2x80x83xe2x80x83(5)
CH3COOxe2x88x92+8FeOOH+6HCO3xe2x88x92+7H+xe2x86x928FeCO3+12H2Oxe2x80x83xe2x80x83(6)
The present invention is of novel methods and apparatuses for drastically improving the rate and efficiency of in situ denitrification, as discussed in the following sections.
The present invention is of a method of in situ biodenitrification of a contaminated site comprising providing to a contaminated site a phosphorus source, namely a polyphosphateor a trimetaphosphate. In the preferred embodiment, the phosphorus source is provided to a saturated zone or an unsaturated zone and a carbon source is also provided, most preferably acetate.
The invention is also of an in situ biodentrification apparatus comprising at least one extraction well for extracting subsurface water from a contaminated site, a container for mixing nutrients into the extracted water, and an injection well for re-introducing the extracted water to the subsurface. In the preferred embodiment, at least three extraction wells are placed approximately on a circle having the injection well as a center, and a phorphorus source is introduced to the container, preferably a polyphosphate or a trimetaphosphate.
The invention is further of a method of biodentrification of a contaminated site comprising: sampling groundwater of the contaminated site; determining levels of nitrate contamination and potential nitrate consuming bacteria from the sampled groundwater; choosing preferred nutrients to be injected into the contaminated site; configuring size, geometry, and pumping rates for wells to be installed at the site; and installing and operating one or more well clusters at the site until the subsurface is denitrified to a desired level. In the preferred embodiment, one or more of the following steps are additionally employed: determining levels of nutrients, determining levels of dissolved oxygen, determining levels of phosphates, generating reaction curves for predetermined constituents at the site, verifying shape of the reaction curves to determine whether significant chemical reactions other than denitrification are occurring, gathering contaminate concentration and hydrological data at the site, and installing and testing a single well cluster at the site.
A primary object of the present invention is to provide biodenitrification methods and apparatuses for drastically improving the rate and efficiency of the process in contaminated sites.
A primary advantage of the present invention is reduction of plugging of infiltration wells by the use of polyphosphate or trimetaphosphate, preferably trimetaphosphate, as the forms of phosphate for injection.
Other objects, advantages and novel features, and further scope of applicability of the present invention will be set forth in part in the detailed description to follow, taken in conjunction with the accompanying drawings, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.